Fluorescence excitation and detection system and method

ABSTRACT

System including an excitation assembly that is configured to excite a sample with first and second wavelengths during first and second excitation events, respectively, of an illumination/detection cycle. The sample provides emission light that includes different first, second, third, and fourth spectral patterns in response to the first and second wavelengths. The system also includes a first detection camera that detects the first spectral pattern during a first measurement phase of the illumination/detection cycle. The first detection camera detects the third spectral pattern during a second measurement phase of the illumination/detection cycle. The system also includes a second detection camera that detects the second spectral pattern during the first measurement phase. The second detection camera detects the fourth spectral pattern during the second measurement phase.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.13/858,575, filed on Apr. 8, 2013, which is a continuation of U.S.application Ser. No. 12/679,652, filed on Mar. 23, 2010, which is anational stage of International Application No. PCT/US2008/077850, filedSep. 26, 2008, which claims priority to and the benefit of U.S.Provisional Patent Application No. 60/975,939, filed Sep. 28, 2007. Eachof the above applications is hereby incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

Embodiments of the present invention relate to the field of nucleic acidsequencing, and more specifically, embodiments of the present inventionprovide methods, systems and devices that utilize a plurality ofexcitation wavelengths to image multiple emission patterns through anoptical detection path comprised of stationary components.

Numerous recent advances in the study of biology have benefited fromimproved methods for analyzing and sequencing of nucleic acids. Forexample, the Human Genome Project has determined the entire sequence ofthe human genome which is hoped to lead to further discoveries in fieldsranging from treatment of disease to advances in basic science. Devicesfor DNA sequencing based on separation of fragments of differing lengthwere first developed in the 1980s, and have been commercially availablefor a number of years. However, such technology involves runningindividual samples through capillary columns filled with polyacrylamidegels and is thus limited in throughput due to the time taken to run eachsample. A number of new DNA sequencing technologies have recently beenreported that are based on the massively parallel analysis ofunamplified, or amplified single molecules, either in the form of planararrays or on beads.

The methodology used to analyze the sequence of the nucleic acids insuch new sequencing techniques is often based on the detection offluorescent nucleotides or oligonucleotides. The detectioninstrumentation used to read the fluorescence signals on such arrays isusually based on either epifluorescence or total internal reflectionmicroscopy. One detection instrument has been proposed that use anoptical sequencing-by-synthesis (SBS) reader. The SBS reader includes alaser that induces fluorescence from a sample within water channels of aflowcell. The fluorescence is emitted and collected by imaging opticswhich comprise one or more objective lens and tube lens. As thefluorescence travels along an optics path within the imaging optics, butprior to reaching a detection camera, the fluorescence propagatesthrough an interference emission filter. The emission filter has theability to select wavelength bands of interest from the fluorescence andblock other wavelength bands that are associated with noise, such aslaser scatter or the emission from orthogonal fluorophores that emit atdifferent wavelengths.

One conventional approach to performing spectral splitting offluorescence is to use bandpass filters in conjunction with an emissionfilter wheel, where an emission filter wheel is located along theoptical path before each detection camera. The emission filter wheel isa mechanical device that is rotated, under control of a servo motor,until an appropriate filter is placed in the optical path of thefluorescence. However, the use of filter wheels and servo motors in thedetection path is not always desirable. As an example of the use of amechanical filter wheel in operation, a high throughput sequencinginstrument that is capable of capturing an image every few hundredmilliseconds, means that in the course of a single days use, the filterwheel is used hundreds of thousands of times. During operation, thefilter wheel is mechanically rotated between imaging cycles whichintroduces complexity and a filter switching time that reduces theoverall operation rate of the detection system. Also, because the filterwheel is a mechanically moving element, it and other moving elementswill have a limited life span and may introduce error over time as theelements wear. Finally, care is needed to properly align and calibratethe filter wheel.

There is a continuing need for better, more robust, and more economicaldevices and systems for fast reliable sequencing of nucleic acids.Embodiments of the present invention seek to address these needs andoffer other benefits which will be apparent upon examination of thecurrent specification, claims, and figures.

SUMMARY OF THE INVENTION

In accordance with an embodiment of the present invention, a detectionsystem is provided for separately detecting different wavelengths ofemission light emitted from a sample. The system comprises a detectionassembly to receive emission light emitted from the sample. Thedetection assembly includes a multi-band dichroic member and at leastfirst and second detection cameras. The multi-band dichroic member has atransmission/reflection characteristic with at least two transmissiveregions separated from each other along the wavelength spectrum and withat least one reflective region separated from the transmissive regionsalong the wavelength spectrum. The dichroic member transmits emissionlight that aligns with the at least two transmissive regions to thefirst detection camera. The dichroic member reflects emission light thataligns with the reflective region to the second detection camera. Thedichroic member multiplexes the detection of emission light signalswithout the use of a filter wheel in the detection assembly. Thecomponents of the detection assembly remain stationary throughoutimaging of multiple colors, for example therefore allowing the use oftwo detectors to record images of three or more colors without the needfor filter wheels or other moving components.

In accordance with at least one embodiment, the system comprises anexcitation assembly to excite the sample. Optionally, the dichroicmember comprises a single dichroic mirror having an incident surfacewith a transmissive/reflective spectrum comprising first and secondtransmissive regions, where the first transmissive region passesfluorescence emitted in response to a first excitation wavelength, whilethe second transmissive region passes fluorescence emitted in responseto a second excitation wavelength. In accordance with at least oneembodiment, the excitation assembly sequentially generates first andsecond excitation beams of different wavelengths during anillumination/detection cycle of an analysis process. The first andsecond excitation beams are generated repeatedly during consecutiveexcitation events of the analysis process. Each excitation event maycomprise illumination with a single wavelength, or with multiplewavelengths. Optionally, the detection assembly may comprise a dual banddichroic member, at least two detection cameras and at least two bandpass filters aligned between the dual band dichroic member and acorresponding one of the detection cameras. Optionally, the detectioncameras may comprise first and second detection cameras aligned with thedichroic member such that fluorescence transmitted to the dichroicmember impinges on the first detection camera and fluorescence reflectedby the dichroic member impinges on the second detection camera, both inresponse to a single excitation event.

In accordance with an alternative embodiment, a method is provided forseparately detecting fluorescence emitted at different wavelengths froma sample. The method comprises exciting a sample with at least first andsecond excitation wavelengths. The sample has first, second and thirdlabels. Each label emits fluorescence at a different wavelength. Thefirst and second labels are excited by the first excitation wavelengthand the third label is excited by the second excitation wavelength. Themethod further comprises directing the fluorescence emitted from thesample onto a detection assembly. The detection assembly includes amulti-band dichroic member configured to have a transmission/reflectioncharacteristic with at least two transmissive regions separated fromeach other along the wavelength spectrum and with at least onereflective region separated from the transmissive regions along thewavelength spectrum. The method further comprises transmittingfluorescence, that aligns with the transmissive regions, through thedichroic member along a transmissive detection path to a first detectioncamera. The method further comprises reflecting fluorescence, thataligns with the reflective region, from the dichroic member along areflective detection path to a second detection camera and detectingfluorescence at the first and second detection cameras, such that thefirst and second labels are detected simultaneously on different ones ofthe first and second detection cameras, and the first and third labelsare detected on a common one of the first and second detection cameras.

Optionally, the excitation operation may sequentially generate first andsecond excitation beams of different wavelengths during a cycle of ananalysis process, where the first and second excitation beams eachgenerate fluorescence with at least two spectral patterns of interestthat are directed by the dichroic member along the transmissive andreflective detection paths. The dichroic member constitutes a non-movingpart that remains stationary and fixed with respect to the sample andwith respect to the transmissive and reflective detection pathsthroughout the analysis process.

In accordance with an alternative embodiment, an excitation anddetection system is provided for separately detecting differentwavelengths of emission light emitted from a sample. The systemcomprises an excitation assembly to excite a sample sequentially withfirst and second wavelengths during first and second excitation eventsin an illumination/detection cycle, respectively. The sample emitsemission light with first and second spectral patterns in response tothe first and second excitation wavelengths, respectively. A detectioncamera receives and measures at least a portion of the emission lightwith the first spectral pattern during a first measurement phase of theillumination/detection cycle. The detection camera receives and measuresat least a portion of the emission light with the second spectralpattern during a second measurement phase of the illumination/detectioncycle. The detection camera outputs first and second data signalsrepresentative of measured portions of the first and second spectralpatterns.

Optionally, the emission light may represent various types ofluminescent light, such as fluorescence, bioluminescence,electroluminescence, radioluminescence and any other emission lightproduced by a sample, where the emission light generates a plurality ofknown spectral patterns that are separable or distinguishable from oneanother along the wavelength spectrum. Optionally, the excitation sourcemay be omitted entirely, such as when the sample utilizeschemiluminescence or radioluminescence and the like.

Optionally, the labels used in one or more of the systems and methodsdescribed herein may comprise a plurality of labeled nucleotidesincluding at least four labeling dyes that emit unique fluorescencespectral patterns corresponding to the four nucleotides G, T, A and C.In response to a first excitation wavelength, the dichroic member mayreflect a first spectral pattern and transmit a second spectral pattern.In response to the second excitation wavelength, the dichroic member mayreflect a third spectral pattern and transmit a fourth spectral pattern,thus allowing the detection of four colors using two illumination eventsand two detection cameras, with no moving parts in the detectionassembly.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a generalized overview of the components of asequencing system formed in accordance with an embodiment of the presentinvention.

FIG. 2 illustrates a block diagram of an excitation and detection systemformed in accordance with an embodiment of the present invention.

FIG. 3 illustrates a timing diagram for exemplary cycles within ananalysis process in accordance with an embodiment.

FIGS. 4A-4D show exemplary configurations of flowcells used in thesequencing system of FIG. 1.

FIG. 5 illustrates an exemplary group of spectral band patterns foremission light that may be associated with a group of dyes used forlabeling bases of interest in accordance with an embodiment.

FIG. 6 illustrates an exemplary transmission/reflection characteristicthat a dichroic member may be configured to exhibit, along withexemplary fluorescence spectral emission patterns, in accordance with anembodiment.

FIG. 7 illustrates the exemplary transmission/reflection characteristicof Figure S, along with additional exemplary fluorescence spectralemission patterns.

FIG. 8 illustrates exemplary filter characteristics of band pass filtersin accordance with an embodiment, along with exemplary fluorescencespectral emission patterns.

FIG. 9 illustrates exemplary filter characteristics of band pass filtersin accordance with an embodiment, along with exemplary fluorescencespectral emission patterns.

FIG. 10 illustrates exemplary filter characteristics of a band passfilters in accordance with an embodiment, along with exemplaryfluorescence spectral emission patterns.

FIG. 11 illustrates exemplary filter characteristics of a band passfilters in accordance with an embodiment, along with exemplaryfluorescence spectral emission patterns.

FIG. 12 illustrates a filter characteristic, excitation beams andspectral emission patterns formed in accordance with an alternativeembodiment.

FIGS. 13-16 illustrate various optional configurations of cameras, lightsources, and other components formed in accordance with alternativeembodiments.

FIG. 17 illustrates a filter characteristic, excitation beams andspectral emission patterns formed in accordance with an alternativeembodiment.

DETAILED DESCRIPTION

Embodiments of the present invention comprise excitation and detectionsystems and methods for detecting fluorescence emitted from a sample.The sample has a plurality of labels, where each label emitsfluorescence having a corresponding spectral pattern along a wavelengthspectrum (emission spectrum). For example, the systems and methods maybe used to analyze a large number of different nucleic acid sequencesfrom, e.g., clonally amplified single-molecule DNA arrays in flowcells,or from an array of immobilized beads. The systems herein are optionallyuseful in, e.g., sequencing for comparative genomics (such as forgenotyping, SNP discovery, BAC-end sequencing, chromosome breakpointmapping, and whole genome sequence assembly), tracking gene expression,micro RNA sequence analysis, epigenomics (e.g., with methylation mappingDNAsel hypersensitive site mapping or chromatin immunoprecipitation),and aptamer and phage display library characterization. Of course, thoseof skill in the art will readily appreciate that the current inventionis also amenable to use for myriad other sequencing applications. Thesystems herein comprise various combinations of optical, mechanical,fluidic, thermal, electrical, and computing devices/aspects which aredescribed more fully below. Also, even though certain embodiments aredirected towards particular configurations and/or combinations of suchaspects, those of skill in the art will appreciate that not allembodiments necessarily comprise all aspects or particularconfigurations (unless specifically stated to do so).

As used throughout, the term “wavelength” shall not be limited to asingle wavelength unless expressly stated to constitute “a singlewavelength” or “only one wavelength”. Instead, the term “wavelength”shall encompass a narrow range of wavelengths located about a desired ortarget wavelength (e.g., 532 nm±10 nm, 660 nm±15 nm).

The exemplary embodiments described herein are discussed in connectionwith the use of fluorescence as a type of emission light produced by asample. However, the present invention is not limited to systems andmethods that utilize fluorescence. Instead, the emission light mayrepresent various types of luminescent light, other than fluorescence,such as bioluminescence, electroluminescence, radioluminescence and anyother emission light produced by a sample, where the emission lightgenerates a plurality of known spectral patterns that are separable ordistinguishable from one another along the wavelength spectrum. Incertain embodiments, the excitation source may be omitted entirely, suchas when the sample utilizes chemiluminescence or radioluminescence andthe like.

System Overview

FIG. 1 shows an exemplary imaging configuration that utilizes totalinternal reflection fluorescence. By way of example, the system of FIG.1 may be constructed to include various components and assemblies asdescribed in international application publication no. WO 2007/123744,titled “System and Devices for Sequence by Synthesis Analysis”, filedMar. 30, 2007, the complete subject matter of which is incorporatedherein by reference in its entirety. As can be seen in FIG. 1, fluiddelivery module or device 100 directs the flow of reagents (e.g.,fluorescent nucleotides, buffers, enzymes, cleavage reagents, etc.) to(and through) flowcell 110 and waste valve 120. The flow cell 110 mayrepresent a substrate having one or more samples provided on or in thesubstrate. In particular embodiments, the flowcell 110 comprisesclusters of nucleic acid sequences (e.g., of about 200-1000 bases inlength) to be sequenced which are optionally attached to the substrateof the flowcell 110, as well as optionally to other components. Theflowcell 110 may also comprise an array of beads, where each beadoptionally contains multiple copies of a single sequence.

The system also comprises temperature station actuator 130 andheater/cooler 135, which can optionally regulate the temperature ofconditions of the fluids within the flowcell 110. The flowcell 110 ismonitored, and sequencing is tracked, by detection assembly 140 whichcan interact with focusing assembly 150. Excitation assembly 160 (e.g.,one or more excitation lasers within an assembly) acts to illuminatefluorescent sequencing reactions within the flowcell 110 via laserillumination through fiber optic 161 (which can optionally comprise oneor more re-imaging lenses, a fiber optic mounting, etc.). Low watt lamp165 (optional), mirror 180 and reverse dichroic 185 are also presentedin the embodiment shown. Additionally, mounting stage 170, allows forproper alignment and movement of the flowcell 110, temperature actuator130, detection assembly 140, etc. in relation to the various componentsof the system. Focus (z-axis) component 175 can also aid in manipulationand positioning of various components (e.g., a lens objective). Suchcomponents are optionally organized upon a framework and/or enclosedwithin a housing structure. It will be appreciated that theillustrations herein are of exemplary embodiments and are notnecessarily to be taken as limiting. Thus, for example, differentembodiments can comprise different placement of components relative toone another (e.g., embodiment A comprises a heater/cooler as in FIG. 1,while embodiment B comprises a heater/cooler component beneath itsflowcell, etc.).

Certain embodiments utilizes sequencing-by-synthesis (SBS). In SBS, aplurality of fluorescently labeled modified nucleotides are used tosequence dense clusters of amplified DNA (possibly millions of clusters)present on the surface of a substrate (e.g., a flowcell).

In particular uses of the systems/devices herein the flowcells 110,containing the nucleic acid samples for sequencing, are placed withinthe appropriate flowcell holder. The samples for sequencing can take theform of single molecules, amplified single molecules in the form ofclusters, or beads comprising molecules of nucleic acid. The nucleicacids are prepared such that they comprise an oligonucleotide primeradjacent to an unknown target sequence. To initiate the first SBSsequencing cycle, one or more differently labeled nucleotides, and DNApolymerase, etc., are flowed into/through the flowcell by the fluid flowsubsystem (various embodiments of which are described herein). Either asingle nucleotide can be added at a time, or the nucleotides used in thesequencing procedure can be specially designed to possess a reversibletermination property, thus allowing each cycle of the sequencingreaction to occur simultaneously in the presence of all four labelednucleotides (A, C, T, G). Where the four nucleotides are mixed together,the polymerase is able to select the correct base to incorporate andeach sequence is extended by a single base.

The heating/cooling components of the system regulate the reactionconditions within the flowcell channels and reagent storageareas/containers (and optionally the camera, optics, and/or othercomponents), while the fluid flow components allow the substrate surfaceto be exposed to suitable reagents for incorporation (e.g., theappropriate fluorescently labeled nucleotides to be incorporated) whileunincorporated reagents are rinsed away. During laser excitation by theexcitation assembly 160, the image/location of emitted fluorescence fromthe nucleic acids on the substrate is captured by the detection assembly140, thereby, recording the identity, in the computer component, of thefirst base for each single molecule, cluster or bead.

The various embodiments of the present invention present several novelfeatures (again, it will be appreciated that not all features arepresent in all embodiments). For example, the systems herein can use twoor more excitation lasers coupled through one or more fiberoptic devicesto illuminate a common area (i.e. the illuminated areas, or footprints,of the lasers at least partially overlap). Additionally, embodiments maycontain a shaking, squeezed, or waveplate modulated fiber (modescrambler) such that the optical intensity from a multimode beam is madeuniform over the whole illumination footprint. The shape of the fiber161 may be adjusted, for example to be square or rectangular, such thatthe shape of the illumination can be matched to the shape of the datacollection device (e.g., a CCD with square pixels). Also, in certainembodiments, a single laser excites two fluorophores, which are detectedusing different emission filters, one with a narrow band emission filternear the laser wavelength, and one with a wider band emission filter ata wavelength longer than the laser wavelength. Such arrangement may‘normalize’ the relative intensities of the two dyes (with the samebandwidth filters, the dye further from the laser wavelength may beweaker due to its lower level of off-wavelength excitation). Theembodiments herein also can comprise a moving stage such that thechemistry (which utilizes cycles of heating and cooling) can happen onthe same instrument, but out of the optical train. The systems hereinmay also contain an autofocus system to allow automated imaging of manytiles, and contain a fluidics system for performing on-line fluidicchanges. The individual components of the system/device (e.g., lightsource, camera, etc.) can optionally each have its own power source orsupply or can optionally all be powered via one source. As will beappreciated, while the components herein are often described inisolation or in relation to only one or two other components, thevarious components in the embodiments are typically operably and/orfunctionally connected and work together in the systems/devices herein.

Flowcells

In various embodiments, the systems and methods herein comprise one ormore substrates upon which the nucleic acids to be sequenced are bound,attached or associated. In certain embodiments, the substrate is withina channel or other area as part of a “flowcell.” The flowcells used inthe various embodiments can comprise millions of individual nucleic acidclusters, e.g., about 2-8 million clusters per channel. Each of suchclusters can give read lengths of at least 25 bases for DNA sequencingand 20 bases for gene expression analysis. The systems and methodsherein can generate over a gigabase (one billion bases) of sequence perrun (e.g., 5 million nucleic acid clusters per channel, 8 channels perflowcell, 25 bases per polynucleotide).

FIGS. 4A and 4B display one exemplary embodiment of a flowcell. As canbe seen, the particular flowcell embodiment, flowcell 400, comprisesbase layer 410 (e.g., of borosilicate glass 1000 μm in depth), channellayer 420 (e.g., of etched silicon 100 μm in depth) overlaid upon thebase layer, and cover, or top, layer 430 (e.g., 300 μm in depth). Whenthe layers are assembled together, enclosed channels are formed havinginlets/outlets at either end through the cover. As will be apparent fromthe description of additional embodiments below, some flowcells cancomprise openings for the channels on the bottom of the flowcell.

It will be appreciated that while particular flowcell configurations arepresent herein, such configurations should not necessarily be taken aslimiting. Thus, for example, various flowcells herein can comprisedifferent numbers of channels (e.g., 1 channel, 2 or more channels, 4 ormore channels, or 6, 8, 10, 16 or more channels, etc.). Additionally,various flowcells can comprise channels of different depths and/orwidths (different both between channels in different flowcells anddifferent between channels within the same flowcell). For example, whilethe channels formed in the cell in FIG. 4B are 100 μm deep, otherembodiments can optionally comprise channels of greater depth (e.g., 500μm) or lesser depth (e.g., 50 μm). Additional exemplary flowcell designsare shown in FIGS. 4C and 4D (for example, a flowcell with “wide”channels, such as channels 440 in FIG. 4C, may have two channels with 8inlet and outlet ports (ports 445—8 inlet and 8 outlet) to maintain flowuniformity and a center wall, such as wall 450, for added structuralsupport. As another example, the flow cell may have offset channels,such as the 16 offset channels (channels 480).

Excitation and Detection Assembly

In certain embodiments herein, the incorporation of specific nucleicacid bases with their accompanying specific fluorescences is tracked viasource excitation and camera observation. In various embodiments,illumination is performed using Total Internal Reflection (TIR)comprising a laser component. It will be appreciated that a “TIRFlaser,” “TIRF laser system,” “TIR laser,” and other similar terminologyherein refers to a TIRF (Total Internal Reflection Fluorescence) baseddetection instrument/system using excitation, e.g., lasers or othertypes of non-laser excitation from such light sources as LED, halogen,and xenon or mercury arc lamps (all of which are also included in thecurrent description of TIRF, TIRF laser, TIRF laser system, etc.herein). Thus, a “TIRF laser” is a laser used with a TIRF system, whilea “TIRF laser system” is a TIRF system using a laser, etc. Again,however, the systems herein (even when described in terms of havinglaser usage, etc.) should also be understood to include thosesystems/instruments comprising non-laser based excitation sources. Insome embodiments, the laser comprises dual individually modulated 50 mWto 500 mW solid state and/or semiconductor lasers coupled to a TIRFprism, optionally with excitation wavelengths of 532 nm and 660 nm. Thecoupling of the laser into the instrument can be via an optical fiber tohelp ensure that the footprints of the two lasers are focused on thesame or common area of the substrate (i.e., overlap).

In certain embodiments, the systems and methods herein comprisecomponent(s) to produce a “top-hat” illumination, e.g., a uniform orsubstantially uniform illumination over a particular illuminationfootprint. Some embodiments comprise one or more aspects thatdynamically change the index of refraction within the mediumtransmitting the illumination (e.g., a fiber) at one or more nodes. Forexample, a fiber can be squeezed at various locations along its lengthto induce a continuously changing index of refraction. Such squeezing ofthe fiber, e.g., a Step Index Fiber, can be used to spatially/temporallyscramble the modes in the fiber to cause sufficient overlap over adesired integration time of the output illumination. The fiber can alsobe shaken, rotated, vibrated or physically deformed in other ways tochange the optical path through the fiber.

In general, the dynamic scrambling of the modes in the fibers allowsachievement of spatially uniform illumination over a minimum userdefined integration time. This thus prevents interference of propagatingmodes of monochromatic light in multimode fibers which would producelight and dark patterns in the resulting beam. It is optionallysufficient that these modes disappear over the minimum integration time.Thus, in some embodiments, the relative path lengths of these modeswithin the illumination beam are rapidly varied by introducing timevariable curvature and index variations into the fiber, e.g., bymechanical means.

It will be appreciated that several parameters of the dynamic modescrambling can optionally be varied or can comprise a range of differentconfigurations. However, in general, dynamic mode scrambling comprisesone or more aspects/components used to dynamically change the index ofrefraction of an illumination beam in order to average out an endillumination footprint.

FIG. 2 illustrates an excitation and detection system 202 that is formedin accordance with an embodiment of the present invention. Theexcitation and detection system 202 generally includes an excitationassembly 204, an autofocus (AF) module 218 and a detection assembly 220.The excitation assembly 204 (corresponding to excitation assembly 160 inFIG. 1) is optically coupled to a sample 212 that is, in turn, opticallycoupled to the AF module 218 and the detection assembly 220(corresponding to the detection assembly 140 in FIG. 1). The sample 212is provided on a substrate 213, such as described in connection withFIGS. 1 and 4A-4C. For example, the sample 212 may represent a pluralityof nucleic acid clusters/beads, with multiple fluorescent labels, whichare attached to a surface of the substrate 213 (e.g., a flow cell). Theexcitation and detection assemblies 204 and 220 can provide a full fieldof view examination of the entire sample 212. For example, theexcitation assembly 204 illuminates the entire surface or active area ofeach tile of the substrate within the flow cell 110 each time anexcitation or illumination event occurs. Illumination of multipleconsecutive tiles allows for large areas of the substrate to be imaged.The excitation assembly 204 illuminates the same or common active area,or tile, in a temporally multiplexed manner with one or more differentexcitation wavelengths during successive excitation events. Theexcitation assembly 204 performs temporal multiplexing by generating oneor more excitation wavelengths sequentially, such as through the use ofmultiple alternating sources or lasers 206 and 208, or multipleexposures of the same lasers. The lasers 206 and 208 are coupled throughan excitation light guide 210 to illuminate a common area, or tile, onthe substrate 213 and sample 212. In response thereto, the sample 212emits fluorescence which is collected by an objective lens 223.

Optionally, the light guide 210 (e.g., separate fiber optics) may beomitted or separate light guides 210 may be used based on the number,type and arrangement of sources or lasers 206 and 208. Optionally, morethan two excitation wavelengths (e.g., 204 or 208) may be generatedsuccessively, such as by providing more than two lasers. Alternatively,a single laser may be used, but controlled to generate the desirednumber of multiple excitation wavelengths. As a further option, aplurality of excitation wavelengths may be generated using one or morelasers, while the number of lasers N and the number of excitationwavelengths M may differ (e.g., M<N). Optionally, the number of lasersmay differ from the number of excitation events such as when multiplelasers are used simultaneously, or when a single laser is used multipletimes. The AF module 218 includes a laser light source that generates afocusing beam 246. The focusing beam 246 is reflected by the dichroicmirror 216 onto the sample 212. The focusing beam 246 is then scatteredand reflected from the sample 212. The scattered light, resulting fromfocusing beam 246, is collected by the objective lens 223. The scatteredlight, resulting from the focusing beam 246, propagates through thedetection assembly 220 and is detected by one or more of detectioncameras 236 and 238. The scattered light then forms a basis forcontrolling focus, as described in more detail in internationalapplication publication no. WO 2003/060589, the contents of which areincluded herein by reference in their entirety.

The control module 211 is electrically connected to the excitationassembly 204 and controls activation and deactivation of the lasers 206and 208 during excitation events. In the example of FIG. 2, a dashedline generally denoted at 214 illustrates an excitation beam that ischanneled from the laser 206, through the light guide 210 and onto thesample 212 at a desired angle of incidence with respect to the surfaceor a reference plane on or within the substrate 213 holding the sample212. A dashed line generally denoted at 215 illustrates an excitationbeam that is channeled from the laser 208, through the light guide 210and onto the sample 212 at a desired angle of incidence with respect tothe surface or a reference plane on or within the substrate holding thesample 212. The control module 211 controls the excitation assembly 204to generate an excitation light pattern throughout a sequence bysynthesis analysis process. By way of example, the control module 211may instruct the lasers 206 and 208 to generate excitation light atsuccessive, non-overlapping periods of time. The laser 206 may supply afirst pulse or burst of light as excitation beam 214 (e.g., at 532 nm)for a predetermined pulse duration, terminate the excitation beam 214,after which the laser 208 may supply a second pulse or burst of light asexcitation beam 215 for a pulse duration and then terminate theexcitation beam 215. In order to record two fluorophores with differentwavelength emissions, each laser may be used once, or more than once ona single area (tile). For example, the sequence to record four differentimages in a single substrate tile may be a: wavelength one; filter one,b: wavelength one; filter two; c: wavelength two; filter three; d:wavelength two; filter four. The exposure time may be the same for eachwavelength emission channel, or may be altered to control the intensityof the fluorescent signal recorded in the different channels. Theexposure time may be the same for every cycle of sequencing, or may beincreased throughout the sequencing run to compensate for anydiminishing of the signal intensity as the cycles are performed.

FIG. 3 illustrates a portion of a sequence analysis process 310, wherethe horizontal axis denotes time. The analysis process 310 may bedivided into illumination/detection cycles 311, where a series ofoperations are repeated during successive portions of each cycle 311.For example, each cycle 311 may include i) an excitation event 316, ii)a measurement phase 317, iii) an excitation event 318 and iv) ameasurement phase 319. Multiple operations may be performedsimultaneously and/or during partially or entirely overlapping timeperiods. For example, the excitation event 316 and measurement phase 317may begin at substantially the same time and end at the same ordifferent times. Similarly, the excitation event 318 and measurementphase 319 may begin at substantially the same time and end at the sameor different times. The excitation event may be at least as long as themeasurement event, but the sample may be illuminated for longer than thetime takes to record an image on the detector. The exposure time foreach tile typically ranges from 50 milliseconds-1 second, and it isunderstood that an excitation beam 214 or 215 may remain active forlonger than an individual measurement event. The control module 211controls both the laser sources 206 and 208, as well as cameras 236 and238, so an exemplary series of control commands may be as follows; beginexcitation, begin camera exposure, end camera exposure, end excitation.During the excitation events 316 and 318, the excitation assembly 204optionally generates excitation beams 314 and 315, consecutively. Duringat least a portion of the measurement phases 317 and 319, the detectionassembly 220 detects and records an amount and spectral pattern offluorescence emitted by the sample 212. It is recognized that theduration of the measurement phases 317 and 319 is not necessarilyco-extensive with the duration of the time period during which thesample 212 emits fluorescence. Instead, the sample 212 may emitfluorescence only during a short initial portion of the measurementphase 317 and 319, which corresponds to an initial portion of theexcitation event 316 and 318.

It is recognized that the durations of the excitation events andmeasurement phases 316 to 319 are not illustrated to scale in FIG. 3.Instead, the durations of the excitation events 316 and 318 may belonger or shorter in relation to the durations of the measurement phases317 and 319. As illustrated, the excitation events and measurementphases 316 to 319 overlap, such that the detection assembly 220 detectsand measures fluorescence during the excitation event 316 and 318, whilea corresponding one of the excitation beams 214 and 215 is beinggenerated.

In the exemplary analysis process of FIG. 3, laser bursts 314 and 315are delivered by the first and second excitation beams 214 and 215successively, and in an interleaved manner, during the excitation events316 and 318 repeatedly, during each cycle 311 throughout the analysisprocess 310. Each individual laser burst 314 and 315 has a laser burstduration 324 and 325, respectively, and amplitude 334 and 335. Leadingedges 344 and 345 of laser bursts 314 and 315 are spaced apart toprovide an inactive inter-burst interval 354 following each laser burst314 before the next laser burst 315, and to provide an inactiveinter-burst interval 355 following each laser burst 315 before the nextlaser burst 314. It is recognized that the burst durations 324 and 325,amplitudes 334 and 335, inter-burst intervals 354 and 355 are notillustrated to scale. Instead, the burst durations 324 and 325 may bevery short, such as an impulse, as compared to the inter-burst intervals354 and 355, while the amplitudes 334 and 335 may be quite substantial.Optionally, the inter-burst intervals 354 and 355 may be substantiallyzero such that the laser bursts 314 and 315 are continuous.

In the example of FIG. 3, the excitation events 316 and 318 arecontrolled to be non-overlapping temporally. The inter-excitationintervals 354 and 355 may be zero, the same or may differ from oneanother in duration. The inter-excitation interval 354 and 355 mayremain constant through an analysis process 310 or may be varied fromcycle 311 to cycle 311 throughout the analysis process 310. Theexcitation durations 324 and 325, are shown in FIG. 3 to be equal, butthe burst duration 324 may vary from the excitation duration 325.Optionally, the excitation durations 324 and 325 may remain constantthroughout an analysis process 310 or may be varied, such that thedurations 324 and 325 during one cycle 311 of an analysis process 310differ from the durations 324 and 325 during a later cycle 311 of theanalysis process 310. The amplitudes 334 and 335 are shown FIG. 3 to beequal, but the amplitude 334 may vary from the amplitude 335.Optionally, the durations 324 and 325 may remain constant through ananalysis process 310 or may be varied also between cycles 311. Also, thedurations 324 and 325 may not equal the duration of the measurementphases 317 and 319. Instead, the laser bursts 314 and 315 may initiatebefore the beginning and terminate after the end of the measurementphases 317 and 319.

In the example of FIG. 2, the lasers 206 and 208 generate excitationlight at different wavelengths that are chosen based on the wavelengthspectrum of the fluorescent bases of interest that will potentially bepresent in the sample 212. In general, a number of bases may be labeledwith a plurality of dyes or combinations of dyes, where each dye emits acorresponding known unique spectral pattern when illuminated withexcitation light at a predetermined wavelength. For example, a number ofbases (e.g., one or more) may be used that are each labeled with one ormore dyes, where the dyes produce spectral patterns that are separatelydistinguishable along the wavelength spectrum. In a particularembodiment of the invention, each of the four bases is labeled with anindividual fluorophore, such that the four bases can be spectrallydistinguished, for example as described in international applicationpublication no. WO 2007/135368, the contents of which are incorporatedherein by reference in their entirety.

FIG. 5 illustrates an exemplary group of spectral patterns 502-505 foremission light (e.g., fluorescence, luminescence, chemiluminescence,etc.) that may be associated with a group of dyes used for labelingbases of interest. In FIG. 5, the horizontal axis plots wavelength andthe vertical axis plots transmission amplitude and transmittance on anormalized scale of 0 to 1. The spectral patterns 502-505 of emissionlight correspond to different dyes. Each individual excitation beamproduces multiple spectral patterns of interest. For example, thefluorescent nucleotides G & T simultaneously emit fluorescence havingspectral patterns 502 and 503 when illuminated with a single excitationbeam having predetermined wavelengths (e.g., 532 nm), while fluorescentnucleotides A & C simultaneously emit fluorescence having spectralpatterns 504 and 505 when illuminated with a single excitation beamhaving one or more different predetermined wavelengths (e.g., 660 nm).The wavelength(s) of the light used to excite dyes A & C differ from thewavelengths of the light used to excite dyes G & T.

The exemplary fluorescent nucleotides are denoted by the letters G, T, A& C. These letters correspond to the nucleotide bases attached to thefluorophores rather than the fluorophores themselves, and there is nosignificance in the order G, T, A, C. Any of the four fluorophores canbe attached to any of the four bases within the scope of the invention.Each spectral pattern 502-505 includes a leading edge 512-515, a peak522-525, a main body portion 532-535 and a tail portion 542-545,respectively. As shown in FIG. 5, while the tail portions 532-535partially overlap one another, the peaks 522-525 are located at separateand discrete wavelengths along the wavelength spectrum, and the mainbody portions 532-535 encompass distributed substantiallynon-overlapping segments of the wavelength spectrum.

Returning to FIG. 2, downstream from the sample 212, a dichroic mirror216 is located to receive the emission light 244 (e.g., fluorescence,luminescence, chemiluminescence, etc.) that is generated at the sample212, such in response to the excitation beams 214 and 215, or inresponse to a chemical reaction when no excitation beams are used. Theemission light 244 is comprised of multiple spectral bands denoted at247-248. The spectral bands 247-248 generally differ from one anotherand may have different center wavelengths, mean wavelengths, medianwavelengths, band widths, shapes and the like. The detection assembly220 is located downstream of the dichroic mirror 216. The detectionassembly 220 provides full field of view detection for the entire areaof each tile of the substrate 212 measured by the objective lens 223.

The detection assembly 220 may include a further focusing component 224,a dichroic member 225, band pass filters 232 and 234, detection cameras236 and 238, and a read out module 237. The focusing component 224 mayfor example be a tube lens, which allows the objective lens 223 to beinfinity corrected. The detection assembly 220 is constructed entirelyof non-moving parts that remain stationary and fixed with respect to oneanother, with respect to an axis of the optical system from theobjective 223 and with respect to reflective and transmissive detectionpaths of the spectral bands 248 and 247, respectively.

In the example of FIG. 2, the spectral bands 247 and 248 correspond tofluorescence emitted by the sample 212 in response to excitation beams214 and 215. The spectral bands 247 and 248 have different spectralpatterns based upon the current dyes that are present on the substrate,and the excitation beam 214 or 215. By way of example, the spectralbands 247 and 248 may correspond to spectral patterns 502 and 503 (FIG.5) when excitation beam 214 impinges upon the substrate 213.Alternatively, the spectral bands 247 and 248 may correspond to spectralpatterns 504 and 505 (FIG. 5) when excitation beam 215 impinges upon thesubstrate 213. Spectral bands 247 and 248 are passed through theoptional dichroic mirror 216 and into the detection assembly 220. Theoptional dichroic mirror 216 allows an autofocus module 218 to beentered into the optical system. Optionally, the autofocus module 218and dichroic 216 may be removed entirely. Optionally, a filter 221(e.g., band pass filter, notch filter, etc.) may be provided after theobjective lens 223, in the space before the tube lens 224. The filter221 blocks out excitation wavelengths. Optionally, the filter 221 may beprovided further downstream (e.g., after the tube lens 224) or removedentirely.

In the example of FIG. 2, the optical component 222 constitutes a tubelens 224, although alternative structures may be used in combinationwith, or in place of, the tube lens 224. The tube lens 224 convergescollimated emission light 244. The detection assembly 220 includes adichroic member 225, such as a dichroic mirror 226, located downstreamof the tube lens 224. The dichroic member 225 of FIG. 2 is formedentirely from a single dichroic mirror 226 having only a single incidentsurface that is coated to include multiple transmission or pass bands(e.g., at least two). The incident surface is coated to provide atransmissive/reflective spectrum comprising a desired combination oftransmissive regions and reflective regions. The transmissive regionsand reflective regions are separate and distinct from one another alongthe wavelength spectrum. Adjacent transmissive and reflective regionsare immediately adjacent one another. The single incident surface of thedichroic mirror 226 may be oriented to form any desired angle ofincidence with respect to an axis of the incoming emission light 244,such as for example 20-45°, or another suitable angle of incidence. Theincident surface has a single common active area that receives bothspectral bands 247 and 248. Fluorescence having spectral patterns 502and 503 and fluorescence having spectral patterns 504 and 505 allimpinge upon the common active area of the incident surface of thedichroic mirror 226. The dichroic mirror 226 reflects spectral band 248and passes spectral band 247 there through. The passed spectral band 247is directed along a transmissive detection path onto a band pass filter232, while the spectral band 248 is reflected along a reflectivedetection path onto a band pass filter 234.

Optionally, the dichroic member 225 may comprise multiple dichroicmirrors or equivalent structures arranged along the optical path andconfigured to provide a desired number of transmission or pass bands.Optionally, the dichroic member 225 may be moved upstream of the tubelens 224 and multiple separate tube lenses 224 may direct light onto thecorresponding detection cameras 236 and 238. The band pass filters 232and 234 block high and low spectral content of the incoming spectralbands 247 and 248, respectively, and pass the portions of the spectralbands 247 and 248 within the upper and lower limits of the pass bands.The limits of the pass bands may be set to sharpen edges of spectralpatterns, block noise, block scatter, block excitation light and thelike. Optionally, the band pass filters 232 and 234 may be removedentirely and replaced with an appropriate filter 221. The passedportions of the spectral bands 247 and 248 are directed ontocorresponding detection cameras 236 and 238.

The band pass filters 232 and 234, and detection cameras 236 and 238 maybe oriented at various angles of incidence with respect to thetransmissive and reflective paths and with respect to one another. Forexample, the detection cameras 236 and 238 may be oriented in aperpendicular geometry or acute angular relation with one another (e.g.,90°, etc.).

The detection cameras 236 and 238 detect the spectral bands 247 and 248,respectively, and provide electrical detection signals 241 and 243 to areadout module 237, for example a computer. The electrical signals canbe provided to the readout module 237 continuously or at discrete timesduring the measurement phases 317 and 319 (FIG. 3). The electricaldetection signals 241 and 243 may be analog or digital signalsrepresenting an amount of emission energy (fluorescent or otherwise)measured by the detection cameras 236 and 238. The detection cameras 236and 238 may output the detection signals 241 and 243 as continuoussignals representative of an instantaneous measurement. Alternatively,the detection cameras 236 and 238 may output the detection signals 241and 243 as a series of period signals representative of discretemeasurements taken at discrete times. The readout module 237 records thedetection signals 241 and 243 and provides a series of images 239representative of the emission light that was detected by each of thedetection cameras 236 and 238. For example, the readout module 237 mayprovide a single image for each detection camera 236 and 238 during eachmeasurement phase. Alternatively, the readout module 237 may provide aseries of images for each detection camera 236 and 238 during eachmeasurement phase.

Next, the operation of the excitation and detection system 202 isdescribed, with respect to FIGS. 6 and 7, in connection with anexemplary cycle of an analysis process. For the example of FIGS. 6 and7, it is assumed that the sample 212 includes one or more bases withfluorescent nucleotides G, T, A & C that emit fluorescence having thespectral patterns 502-505 illustrated in FIG. 5.

FIGS. 6 and 7 illustrate an exemplary transmission/reflectioncharacteristic that the dichroic member 225 may be configured toexhibit, along with the spectral patterns 502-505 of FIG. 5. Thedichroic member 225 may be configured to exhibit differenttransmission/reflection characteristics based on the expected spectralemission patterns of other dyes that may be used. The horizontal axes inFIGS. 6 and 7 represent wavelength, while the vertical axes representemission or transmissivity. As shown in each of FIGS. 6 and 7, thedichroic member 225 is constructed with at least two pass bands 710 and712. The pass band 710 includes cutoffs at transmittance lower and upperlimits 714 and 716, while the pass band 712 includes lower and upperlimits 718 and 720. The pass bands 710 and 712 are separated from oneanother along the wavelength spectrum such that the upper limit 716 ofthe lower pass band 710 is spaced apart by a desired wavelength range(denoted at 717) below the lower limit 718 of the upper pass band 712.The dichroic member 225 exhibits at least a pair of transmissive regions722 and 724 that are separated by a reflective region 728 and bound byouter reflective regions 726 and 730. Fluorescent incident light, havingwavelengths within the transmissive regions 722 and 724, is passedthrough the dichroic member 225 onto band pass filter 232 and detectioncamera 236. Incident light, having wavelengths within the reflectiveregions 726, 728 and 730, is reflected by the dichroic member 225 ontoband pass filter 234 and detection camera 238, and hence the two signalsare recorded simultaneously.

FIG. 6 corresponds to the measurement phase associated with a firstexcitation event when the laser 206 generates the excitation beam 214.FIG. 7 corresponds to the measurement phase associated with a secondexcitation event when the laser 208 generates the excitation beam 215.During the first excitation event, fluorescence is generated having thespectral patterns 502 and 503. During the second excitation event,fluorescence is generated having the spectral patterns 504 and 505.

Although the example is shown with two consecutive excitations withdifferent wavelengths, the use of the dual band pass dichroic means thatboth excitation events can be performed simultaneously, using both beams214 and 215 at the same time. In this embodiment, the optical system maycomprise four detection cameras rather than the two shown in FIG. 2,although the concept of using two cameras and two excitation wavelengthsis within the scope of the invention. For the consecutive excitationevents, the order of wavelength illuminations is not relevant, and thelaser sources may be used in any order; e.g., spectral patterns 504 and505 may be generated either before or after spectral patterns 502 and503.

Following initiation of excitation beam 214, the sample (when containingfluorescent nucleotides G, T, A and C discussed above) emitsfluorescence having the spectral patterns 502 and 503 (as shown in FIG.6). In the case where wavelengths 214 and 215 are used simultaneously,spectral patterns 502-505 are generated simultaneously. The portion ofthe spectral pattern 502 (shown associated with dye G) that falls withinthe reflective region 726 is reflected by the dichroic mirror 226 ontothe band pass filter 234. The portion of the spectral pattern 503 (shownassociated with dye T) that falls within the region 722 is passedthrough the dichroic mirror 226 and directed onto the band pass filter232. As is evident in FIG. 6, the amount of energy associated with theleading edge 513 of the spectral pattern 503 that falls within thereflective region 726 is relatively small in comparison to the amount ofenergy within the main body portion 532 of the spectral pattern 502 thatis reflected by the dichroic mirror 226. Hence, the majority of thesignal detected by camera 238 corresponds to spectral pattern 502 (the‘G’ signal), and the leading edge 513 does not detrimentally impact theaccuracy of the detection camera 238.

The signal detected by camera 236 will comprise components deriving fromboth tail portion 542 of spectral pattern 502, and the main body portion533 of spectral pattern 503. The tail portion 542 and main body portion533 may be of similar intensity without compromising the accuracy ofdetermining whether the identity of the object is ‘T’ or ‘G’, due to thedetection of all objects of signal ‘G’ on camera 236.

The leading edge 513 and the tail portion 543 of the spectral pattern503 fall within reflective regions 726 and 728, respectively, and thusare reflected by the dichroic mirror 226. However, as explained above,the amount of energy associated with the leading edge 513 is relativelysmall in comparison to the amount of energy within the main body portion532 of the spectral pattern 502 that is reflected by the dichroic mirror226. The tail portion 543 is removed using the band pass filter 234, andhence, the leading edge 513 and tail portion 543 do not detrimentallyimpact the accuracy of the detection camera 238. The dichroic mirror 226lets through all the light in pass bands 710 and 712. As an option, theband pass filters 232 and 234 may be configured to block partially theunwanted leading edges 513 and 515 and tail portions 543 and 545, beforereaching the detection cameras 236 and 238.

Turning to FIG. 7, following initiation of the excitation beam 215, thesample 212 emits fluorescence having the spectral patterns 504 and 505(as shown in FIG. 7). The main body portion 534 of the spectral pattern504 (shown associated with dye A) that falls within the reflectiveregion 728 is reflected by the dichroic mirror 226 onto the band passfilter 234. The main body portion 535 of the spectral pattern 505 (shownassociated with dye C) that falls within the transmissive region 724 ispassed through the dichroic mirror 226 and directed onto the band passfilter 232. As is evident in FIG. 7, the amount of energy associatedwith the leading edge 515 of the spectral pattern 505 that falls withinthe reflective region 728 is relatively small in comparison to theamount of energy within the main body portion 534 of the spectralpattern 504 that is reflected by the dichroic mirror 226. Hence, theleading edge 515 does not detrimentally impact the accuracy of thedetection camera 238.

The signal detected by camera 236 will comprise components deriving fromboth the main body portion 535 of spectral pattern 505, and the tailportion 544 of spectral pattern 504. The main body portion 535 and tailportion 544 may be of similar intensity without compromising theaccuracy of determining whether the identity of the object is ‘A’ or‘C’, due to the detection of all objects of signal ‘A’ on camera 236.

Similarly, it is apparent that the leading edge 515 and the tail portion545 of the spectral pattern 505 falls within reflective regions 728 and730, respectively, and thus are reflected by the dichroic mirror 226.However, as explained above, the amount of energy associated with theleading edge 515 and tail portion 545 is relatively small in comparisonto the amount of energy within the main body portion 534 of the spectralpattern 504 that is reflected by the dichroic mirror 226. The tailportion 545 is removed using the band pass filter 234, and hence, theleading edge 515 and tail portion 545 do not detrimentally impact theaccuracy of the detection camera 238. The dichroic mirror 226 letsthrough all the light in pass bands 710 and 712. As an option, the bandpass filters 232 and 234 may be configured to block partially theunwanted leading edges 513 and 515 and tail portions 543 and 545, beforereaching the detection cameras 236 and 238.

In the example of FIGS. 6 and 7, the lower limits 714 and 718 are usedas transmittance cut off frequencies to discriminate between closelyspaced spectral patterns, whereas the upper limits 716 and 720 are not.Optionally, the transmissive regions 722 and 724 could be switched withreflective regions 726 and 728, respectively, to pass spectral patterns502 and 504, and reflect spectral patterns 503 and 505. As a furtheroption, additional spectral patterns may be used such that the lower andupper limits 714, 716, 718 and 720 could all be used to discriminatebetween adjacent spectral patterns (e.g., when 5 or 8 dyes are used togenerate 6 or 8 spectral patterns).

FIGS. 8 and 10 illustrate exemplary filter characteristics that the bandpass filters 232 may be configured to exhibit when used with thespectral emission patterns 502-505 of FIG. 5. FIGS. 9 and 11 illustrateexemplary filter characteristics that the band pass filters 234 may beconfigured to exhibit when used with the spectral emission patterns502-505 of FIG. 5. The horizontal axes in FIGS. 8-11 representwavelength, while the vertical axes represent transmissivity.

As shown in FIGS. 8 and 10, the band pass filter 232 is constructed tohave multiple pass bands 810 and 812. The pass band 810 includes lowerand upper limits 814 and 816, while the pass band 812 includes lower andupper limits 818 and 820. The pass bands 810 and 812 are separated fromone another along the wavelength spectrum such that the upper limit 816of the lower pass band 810 is spaced apart by a desired wavelength range(denoted at 817) below the lower limit 818 of the upper pass band 812.Incident light, having wavelengths within the pass bands 810 and 812, ispassed through the filter 232 onto detection camera 236. Emissionincident light, having wavelengths outside the pass bands 810 and 812,is blocked. Thus, as shown in FIGS. 8 and 10, the majority of theemission light having spectral patterns 503 and 505 are conveyed throughthe filter 232, while the regions of the emission light having spectralpatterns 502 and 504 are blocked.

As shown in FIGS. 9 and 11, the band pass filter 234 is constructed tohave multiple pass bands 910 and 912. The pass band 910 includes lowerand upper limits 914 and 916, while the pass band 912 includes lower andupper limits 918 and 920. The pass bands 910 and 912 are separated fromone another along the wavelength spectrum such that the upper limit 916of the lower pass band 910 is spaced apart by a desired wavelength rangefrom the lower limit 918 of the upper pass band 912. Emission incidentlight, having wavelengths within the pass bands 910 and 912, is passedthrough the filter 234 onto detection camera 238. Emission incidentlight, having wavelengths outside the pass bands 910 and 912, isblocked. Thus, as shown in FIGS. 9 and 11, the majority of thefluorescence having spectral patterns 502 and 504 are conveyed throughthe filter 234, while the majority of the fluorescence having spectralpatterns 503 and 505 are blocked.

In accordance with at least one embodiment described the multi-pass banddichroic member affords the technical effect of separating the spectralpatterns 502 and 503 for delivery to different detection camerasutilizing a detection assembly having non-moving parts that remainstationary and fixed throughout the analysis process. Further, inaccordance with at least one embodiment, the multi-pass band dichroicmember affords the technical effect that spectral patterns 502 and 504may be excited with different excitation beams while being imaged on thesame detection camera.

As explained above, more than two excitation wavelengths may be used.Multiple excitation wavelengths can be present in a single excitationbeam, such as a beam of white light, or each wavelength can be presentin a separate beam, such as a laser beam. It may be equally possible toexcite the multiple fluorophores using a single fixed laser. Suchsystems for exciting multiple fluorophores using a single laser may alsoinclude the use of energy transfer labels from a single donor todifferent acceptors, or the use of labels with different Stokes shifts,such as Quantum dots or similar microparticles.

FIG. 12 illustrates an alternative embodiment in which atransmission/reflection characteristic for a single dichroic mirror 226may be configured for use with four separate excitation wavelengths1206-1209. The excitation wavelengths 1206-1209 produce fluorescencewith the spectral patterns 1202-1205, respectively. The horizontal axisrepresents wavelength, while the vertical axis represents transmittance.The dichroic mirror 226 is constructed with at least two pass bands 1210and 1212. The pass band 1210 includes lower and upper limits 1214 and1216, while the pass band 1212 includes lower and upper limits 1218 and1220. Fluorescence with spectral patterns 1203 and 1205 within thetransmissive regions 1222 and 1224 are passed through the dichroicmirror 226 onto band pass filter 232 and detection camera 236.Fluorescence with spectral patterns 1202 and 1204 within the reflectiveregions 1226 and 1228 are reflected by the dichroic mirror 226 onto bandpass filter 234 and detection camera 238.

The excitation beams 1206-1209 may be produced sequentially or incombinations (e.g., pair 1206 and 1208, then pair 1207 and 1209). Forexample, fluorescence may be generated having the spectral emissionpatterns 1202 and 1203 for a period of time following the excitationbeams 1206 and 1207, but before initiation of the excitation beams 1208and 1209. Fluorescence may be generated having the spectral patterns1204 and 1205 for a period of time following the excitation beams 1208and 1209.

Optionally, the excitation assembly 204 may be controlled to generatemultiple excitation beams simultaneously, where each excitation beam hasa distinct wavelength. As explained above, the labels may be configuredto emit fluorescence with multiple spectral patterns in response to eachwavelength of excitation beam.

FIG. 17 illustrates an alternative embodiment in which atransmission/reflection characteristic for a single dichroic mirror maybe configured for use with four excitation beams 1714-1717 that aregenerated simultaneously. The horizontal axis represents wavelength,while the vertical axis represents transmittance. The excitation beams1714 and 1715 collectively produce fluorescence with the spectralpatterns 1702, 1703 and 1704, respectively. The excitation beams 1716and 1717 collectively produce fluorescence with the spectral patterns1705, 1706 and 1707. In the example of FIG. 17, a group of N excitationwavelengths (e.g., 1714-1715) are used to produce a group of M spectralpatterns (e.g., 1702-1704). The dichroic mirror 226 is constructed withat least three pass bands 1710, 1712 and 1714. Fluorescence withspectral patterns 1703, 1705 and 1707 within the transmissive regions1722, 1724 and 1732 are passed through the dichroic mirror 226 onto bandpass filter 232 and detection camera 236. Fluorescence with spectralpatterns 1702, 1704 and 1706 within the reflective regions 1726, 1728and 1730 are reflected by the dichroic mirror 226 onto band pass filter234 and detection camera 238. The excitation wavelengths 1714 to 1717can be produced, simultaneously, sequentially or in combinations.Optionally, the excitation wavelengths 1715 and 1717 may be removedentirely, and only excitation wavelengths 1714 and 1716 illuminate asample to produce the illustrated spectral patterns 1702 to 1707.

Devices for Detecting Fluorescence

The detection devices 236 and 238 may be, for example photodiodes orcameras. In some embodiments herein, the detection camera can comprise a1 mega pixel CCD-based optical imaging system such as a 1002×1004 CCDcamera with 8 m pixels, which at 20× magnification can optionally imagean area of 0.4×0.4 mm per tile using a laser spot size of 0.5×0.5 mm(e.g., a square spot, or a circle of 0.5 mm diameter, or an ellipticalspot, etc.). The detection cameras can optionally have more or less than1 million pixels, for example a 4 mega pixel camera can be used. In manyembodiments, it is desired that the readout rate of the camera should beas fast as possible, for example the transfer rate can be 10 MHz orhigher, for example 20 or 30 MHz. More pixels generally mean that alarger area of surface, and therefore more sequencing reactions or otheroptically detectable events, can be imaged simultaneously for a singleexposure. In particular embodiments, the CCD camera/TIRF lasers hereinare capable of collecting about 6400 images to interrogate 1600 tiles(since images are optionally done in 4 different colors per cycle usingcombinations of filters, dichroics and detectors as described herein.For a 1 Mega pixel CCD, certain images optionally can contain betweenabout 5,000 to 50,000 randomly spaced unique nucleic acid clusters(i.e., images upon the flowcell surface). At an imaging rate of 2seconds per tile for the four colors, and a density of 25000 clustersper tile, the systems herein can optionally quantify about 45 millionfeatures per hour. At a faster imaging rate, and higher cluster density,the imaging rate can be significantly improved. For example, at themaximum readout rate of a 20 MHz camera, and a resolved cluster every 20pixels, the readout can be 1 million clusters per second. As describedherein, the light can be split to simultaneously image two colors ontotwo cameras, or even four colors onto four cameras. If four cameras areused in parallel, it is thus possible to sequence 1 million bases persecond, or 86.4 billion bases per day.

There are at least two ways of splitting up the optical signals for atwo camera system. If two lasers are used, there may be a red excitationand a green excitation, with half the emission light split towards eachcamera. Alternatively both lasers may be used in both illuminationcycles, and the light may pass through a suitable dichroic mirror 226,so sending the red light in one direction, and the green light in adifferent direction. Such system prevents the signal losses associatedwith beam splitting, but does mean that two of the dyes are exposed tothe laser before their intensity is recorded. In some such embodiments,the excitation blocker can comprise a dual notch filter (e.g., 532 and660 nm). In such an embodiment, band pass filters 232 and 234 aretypically rotated between images in order to measure region 722 during afirst excitation event, and 726 on a second excitation event of the samewavelength onto the same camera. Embodiments of the present inventiondescribed in the current application avoid the use of filter rotation byusing a stationary, dual band pass dichroic member, which means thatrather than performing two consecutive illuminations and measurements inwhich both illuminations require both wavelengths at each illumination,the consecutive illuminations can be performed using a single wavelengthper illumination. In accordance with certain embodiments, the advantagesinclude a reduction in the time a sample is illuminated leading toreduced photobleaching that would otherwise cause the signal of thefluorescent signal to be reduced before the image is recorded. A furtheradvantage is the avoidance of moving parts in the optical detectionsystem.

A “tile” herein is functionally equivalent to the image size mapped ontothe substrate surface. Tiles can be, e.g., 0.33 mm×0.33 mm, 0.5 mm×0.5mm, 1 mm×1 mm, 2 mm×2 mm etc, although the size of the tile will dependto a large extent on the number and size of pixels on the camera and thedesired level of magnification. Also, it will be appreciated that thetile does not have to equal the same size or shape as the illuminationfootprint from the laser (or other light source), although this can beadvantageous if the minimization of photobleaching is desired.

As stated previously, in the various embodiments herein, thecamera/laser systems collect fluorescence from 4 different fluorescentdyes (i.e., one for each nucleotide base type added to the flowcell).

FIGS. 1 and 13-16 show alternative embodiments of the cameras and lasersof the present invention, including a backlight design, a TIRF Imagingconfiguration, a laser focusing configuration, a white-light viewingconfiguration, and an alternative laser focusing design. The white lightexcitation source is optional, and can be used as well as, or insteadof, the excitation lasers. FIG. 1 shows the backlight design systemwhilst recording an image in the TIRF imaging configuration. Theconfiguration in FIG. 1 for the TIRF imaging is optionally aconfiguration of the backlight design set-up shown in FIG. 13. In FIG.1, one of the two lasers (in laser assembly 160) is used to illuminatethe sample (in flowcell 110), and a single one of the four emissionfilters (in filter switching assembly 145) is selected to record asingle emission wavelength and to cut out any stray laser light. Duringimaging, both focus laser (150) and optional white light lamp (165) canbe prevented from illuminating the sample either by being blocked with ashutter or switched off. Laser illumination 101 and illumination fromthe flowcell up through the lens objective and camera 102 are alsoshown. FIG. 13 shows all the components of the system in the backlightdesign but without the specific TIRF imaging configuration. Cf. FIGS. 1and 13. Thus FIG. 13 shows: fluid delivery module 1300, flowcell 1310,waste valve 1320, temperature actuator 1330, heating/cooling component(e.g., Peltier) 1335, camera (e.g., CCD camera) 1340, objective lens1342, filter switching assembly 1345, focusing laser assembly 1350,excitation lasers assembly 1360, low watt lamp 1365, precision XY stage1370, focus (z-axis) device 1375, mirror 1380, “reverse” dichroic 1385,and laser fiber optic 1390.

FIG. 14 shows a similar system as that in FIG. 1, but in the laserfocusing configuration where the excitation lasers (in laser assembly1460) and optional white light 1465 are switched off Focusing laser 1450is on and shines into the system, hits beam splitter 1485 (e.g., apick-off mirror x1% beam splitter) which direct a faint beam 1420 downthe objective to hit a small spot on the sample (in flowcell 1410). Thereflected light from the sample returns up objective (1442) through anempty slot in filter wheel switching assembly 1445 and is imaged by CCDcamera 1440. The position of the spot on the camera is used to ensurethe sample is at the right distance from the objective, and thereforethe image will be in focus. The numbering of the elements in FIG. 14 issimilar to that of the elements in FIG. 13, but numbered as “14” ratherthan “13,” e.g., 1460 corresponds to a similar element as 1360, etc. Inembodiments where the band pass filters are fixed, the autofocus lasermay be a suitable wavelength to pass through one of the filters ratherthan using an empty slot in the filter switching assembly. The smallsize and low intensity of the spot prevents damage to the sample.

FIG. 15 shows the optional white light viewing configuration, wherefocus laser 1550 and illumination lasers 1560 are off. In suchconfiguration the white light from low watt lamp 1565 goes into thesystem as beam 1503 and is imaged directly on the camera. Here too, thenumbering of elements, except for beam 1503, etc., follows that of FIGS.13 and 14. FIG. 16 shows an alternative focus configuration where thesystem contains second focusing camera 1641, which can be a quadrantdetector, PSD, or similar detector to measure the location of thereflected beam reflected from the surface. This configuration allows forfocus control concurrent with data collection. The focus laserwavelength is optionally longer than the reddest dye emission filter.

Illumination Systems

A variety of illumination systems may be used in devices according tothe present invention. The illumination systems can comprise lampsand/or lasers. In particular embodiments, excitation generated from alamp or laser can be optically filtered to select a desired wavelengthfor illumination of a sample. The systems can contain one or moreillumination lasers of different wavelengths. For example the systemsherein may contain two lasers of 532 nm and 660 nm, although lasers withother wavelengths may also be used. Additionally, in variousembodiments, the lasers in the systems herein are actively temperaturecontrolled to 0.1 C, have TTL modulation for the 660 nm laser diode withrise time less than 100 ms; have integrated manual shutters for fastmodulation of the 532 nm laser, have integrated beam shaping optics toensure the optimum beam aspect ratio is maintained at the instrumentinterface to maximize signal to noise ratio, have integrated modescrambler to reduce ripple on the output of the multi-mode fiber, andhave minimal heat generation. The shutters and TTL modulation are usedto ensure that the illumination is only on the sample surface whilst thecamera is recording images. Illumination of fluorophores can causephotobleaching, and therefore exposure of substrates to the laser whennot needed is generally minimized, especially before the images arerecorded.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovemay be used in various combinations. All publications, patents, patentapplications, or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application, orother document were individually indicated to be incorporated byreference for all purposes. It is to be understood that the abovedescription is intended to be illustrative, and not restrictive. Forexample, the above-described embodiments (and/or aspects thereof) may beused in combination with each other. In addition, many modifications maybe made to adapt a particular situation or material to the teachings ofthe invention without departing from its scope. While the dimensions,types of materials and coatings described herein are intended to definethe parameters of the invention, they are by no means limiting and areexemplary embodiments. Many other embodiments will be apparent to thoseof skill in the art upon reviewing the above description. The scope ofthe invention should, therefore, be determined with reference to theappended claims, along with the full scope of equivalents to which suchclaims are entitled. In the appended claims, the terms “including” and“in which” are used as the plain-English equivalents of the respectiveterms “comprising” and “wherein.” Moreover, in the following claims, theterms “first,” “second,” and “third,” etc. are used merely as labels,and are not intended to impose numerical requirements on their objects.Further, the limitations of the following claims are not written inmeans-plus-function format and are not intended to be interpreted basedon 35 U.S.C. §112, sixth paragraph, unless and until such claimlimitations expressly use the phrase “means for” followed by a statementof function void of further structure.

1. (canceled)
 2. A system comprising: an excitation assembly configuredto excite a sample with first and second wavelengths during first andsecond excitation events, respectively, of an illumination/detectioncycle, wherein the sample provides emission light that includesdifferent first, second, third, and fourth spectral patterns in responseto the first and second wavelengths; a first detection camera configuredto detect the first spectral pattern during a first measurement phase ofthe illumination/detection cycle, the first detection camera configuredto detect the third spectral pattern during a second measurement phaseof the illumination/detection cycle; and a second detection cameraconfigured to detect the second spectral pattern during the firstmeasurement phase, the second detection camera configured to detect thefourth spectral pattern during the second measurement phase.
 3. Thesystem of claim 2, wherein the first detection camera and the seconddetection camera concurrently detect the first and second spectralpatterns during the first measurement phase.
 4. The system of claim 2,wherein the first detection camera successively detects the first andthird spectral patterns.
 5. The system of claim 2, further comprising adetection assembly that includes the first and second detection camerasand a dichroic mirror, the dichroic mirror reflecting the first spectralpattern and the third spectral pattern during the first and secondmeasurement phases, respectively, the dichroic mirror permitting thesecond spectral pattern and the fourth spectral pattern to passtherethrough during the first and second measurement phases,respectively.
 6. The system of claim 5, wherein the detection assemblyincludes a band pass filter, the first and third spectral patterns beingdirected along a reflection detection path toward the band pass filterprior to the first detection camera, the band pass filter configured toblock high and low spectral content of at least one of the first orthird spectral patterns.
 7. The system of claim 6, wherein the band passfilter has two pass bands that are separated from each other along awavelength spectrum.
 8. The system of claim 6, wherein the detectionassembly includes another band pass filter, the second and fourthspectral patterns being directed along a transmission detection pathtoward the other band pass filter prior to the second detection camera,the other band pass filter configured to block high and low spectralcontent of at least one of the second or fourth spectral patterns. 9.The system of claim 2, wherein the first and second detection camerasoutput first, second, third and fourth data signals representative ofdetected portions of the first, second, third and fourth spectralpatterns.
 10. The system of claim 9, wherein the illumination/detectioncycle is repeated numerous times.
 11. The system of claim 10, furthercomprising a readout module that receives the first, second, third, andfourth data signals, the readout module configured to provide aplurality of images that are representative of the emission lightdetected by the first and second detection cameras.
 12. A methodcomprising: (a) exciting a sample with a first wavelength during a firstexcitation event, the sample providing first and second spectralpatterns in response to the first wavelength; (b) detecting the firstspectral pattern with a first detection camera; (c) detecting the secondspectral pattern with a second detection camera; (d) exciting the samplewith a second wavelength during a second excitation event, the sampleproviding third and fourth spectral patterns in response to the secondwavelength; (e) detecting the third spectral pattern with the firstdetection camera; and (f) detecting the fourth spectral pattern with thesecond detection camera.
 13. The method of claim 12, wherein detectingthe first spectral pattern and detecting the second spectral patternoccurs concurrently.
 14. The method of claim 12, wherein detecting thefirst spectral pattern and detecting the third spectral pattern occursseparately.
 15. The method of claim 12, wherein the first spectralpattern is reflected by a dichroic member and the second spectralpattern is permitted to pass through the dichroic member and wherein thethird spectral pattern is reflected by the dichroic member and thefourth spectral pattern is permitted to pass through the dichroicmember.
 16. The method of claim 15, further comprising blocking high andlow spectral content of at least one of the first or third spectralpatterns.
 17. The method of claim 16, wherein a band pass filter blocksthe high and the low spectral content, the band pass filter having twopass bands that are separated from each other along a wavelengthspectrum.
 18. The method of claim 12, further comprising outputtingfirst, second, third, and fourth data signals from the first and seconddetection cameras that are representative of detected portions of thefirst, second, third and fourth spectral patterns.
 19. The method ofclaim 19, wherein steps (a)-(f) constitute an illumination/detectioncycle, the method further comprising repeating theillumination/detection cycle numerous times.
 20. The method of claim 20,further comprising providing a plurality of images that arerepresentative of the emission light detected by the first and seconddetection cameras.